Journal: The Journal of Clinical Investigation

Article Title: The CDK4/6-EZH2 pathway is a potential therapeutic target for psoriasis

doi: 10.1172/JCI134217

Figure Lengend Snippet: (A) Human primary keratinocytes were treated for 1 hour with 100 ng/mL IL-36α or 200 ng/mL IL-17A and 10 ng/mL TNF-α. The CDK4/6 inhibitor abemaciclib (Abe) or an ethanol vehicle control (Ctrl) were added in parallel. Phosphorylation of RB (pRB) served as a control for CDK4/6 inhibition, and actin as a loading control. Relative mRNA levels of IκBζ (NFKBIZ) were normalized to the reference gene RPL37A. (B) Luciferase assay of IκBζ (NFKBIZ) promoter activity in HaCaT cells that were cytokine-stimulated for 24 hours in the presence or absence of the CDK4/6 inhibitors abemaciclib or palbociclib (Pal). Relative luciferase (luc) activity was normalized to an internal Renilla luciferase control that was transfected in parallel. Endogenous protein levels were analyzed as input controls by immunoblotting (bottom). (C and D) CDK4 and CDK6 were depleted in primary human keratinocytes by lentiviral transduction of shRNA. Ctrl shRNA– or CDK4/6 shRNA–depleted cells were treated with (C) IL-36α or (D) IL-17A/TNF-α, similar as in A. (E and F) Human primary keratinocytes were stimulated with IL-36α as in A. (E) Cytokine gene expression in CDK4/6 inhibitor–treated cells. (F) Relative gene expression levels in IL-36α–treated control or CDK4/6-depleted cells. (G) Transient overexpression of CDK4, CDK6, or CDK9 in HaCaT cells, treated for 1 hour with 100 ng/mL IL-36α. (H) Cytokine gene expression in IL-36α–treated primary keratinocytes overexpressing IκBζ in the presence or absence of abemaciclib. For all analyses, n = 3 ± SD. Significance was calculated using a 1-way ANOVA for multiple groups and a 2-tailed Student’s t test for comparing 2 groups: *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Recombinant human IL-36α (6995-IL; aa 6-158), IL-36γ (6835-IL; aa 18-169), and mouse IL-36α (7059-ML; aa 6-160) were purchased from R&D Systems.

Techniques: Control, Phospho-proteomics, Inhibition, Luciferase, Activity Assay, Transfection, Western Blot, Transduction, shRNA, Gene Expression, Over Expression

Journal: The Journal of Clinical Investigation

Article Title: The CDK4/6-EZH2 pathway is a potential therapeutic target for psoriasis

doi: 10.1172/JCI134217

Figure Lengend Snippet: (A) Luciferase assay of the NFKBIZ promoter in HEK293T cells after transient expression of CDK4, CDK6, STAT3, or p65, alone or in combination. The plasmid amounts for STAT3 (200 ng) and p65 (70 ng) were adjusted to achieve similar luciferase activity in the absence of CDK4/6 expression. Overexpression of the HA-tagged CDK4 and CDK6 proteins was detected using a HA-antibody. (B) Primary human keratinocytes with a transient overexpression of hyperactive STAT3 (STAT3C) were treated for 1 hour with 100 ng/mL IL-36α and abemaciclib (Abe). NFKBIZ mRNA levels normalized to RPL37A. Immunoblot analysis of STAT3C overexpression and CDK4/6 inhibition. (C) IκBζ target gene expression in STAT3C-overexpressing primary keratinocytes. Treatment as in B. (D) Luciferase activity assay of the NFKBIZ promoter in HEK293T cells overexpressing STAT3 alone or in combination with WT CDK6 (wt), hyperactive CDK6 (S178P), or a kinase-dead CDK6 mutant (CDK6 DN). For all analyses, n = 3 ± SD. Significance was calculated using a 1-way ANOVA for multiple groups and a 2-tailed Student’s t test for comparing 2 groups: *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Recombinant human IL-36α (6995-IL; aa 6-158), IL-36γ (6835-IL; aa 18-169), and mouse IL-36α (7059-ML; aa 6-160) were purchased from R&D Systems.

Techniques: Luciferase, Expressing, Plasmid Preparation, Activity Assay, Over Expression, Western Blot, Inhibition, Targeted Gene Expression, Mutagenesis

Journal: The Journal of Clinical Investigation

Article Title: The CDK4/6-EZH2 pathway is a potential therapeutic target for psoriasis

doi: 10.1172/JCI134217

Figure Lengend Snippet: (A) STAT3 activity was detected by analyzing the phosphorylation state at tyrosine 705 (Y705) and threonine 727 (T727) of STAT3 in primary human keratinocytes. After overnight starvation, cells were stimulated for 1 hour with IL-36α or IL-17A/TNF-α in the presence or absence of abemaciclib (Abe). (B) STAT3 activity in CDK4- and CDK6-depleted keratinocytes. Stimulation as in A. (C) Immunoblot detection of phosphorylated STAT3 (Y705) in IL-36α–stimulated keratinocytes, in which EZH2 function was suppressed by the EZH2 inhibitor EPZ6438 (EPZ, 10 μM) or shRNA-mediated knockdown. Detection of H3K27me3 controlled effective EZH2 inhibition or depletion. (D) Immunoblot detection of phosphorylated EZH2 at threonine 345 (T345) and threonine 487 (T487) in abemaciclib-treated or CDK4/6-depleted keratinocytes following stimulation with IL-36α. (E) Coimmunoprecipitation of EZH2 and STAT3 in HaCaT cells treated for 30 minutes with IL-36α in the presence or absence of abemaciclib. An EZH2-specific antibody or IgG was used for pull down of protein complexes. STAT3 and pEZH2 (T345) were detected by immunoblotting. (F) Luciferase activity assay of the NFKBIZ promoter in HEK293T cells, which transiently overexpress CDK6, WT EZH2 (wt), mutant EZH2 (T345A), or STAT3, alone or in combination. Equal protein expression was detected by immunoblotting. n = 3 ± SD. (G) Gene expression in IL-36α– and abemaciclib-treated, primary keratinocytes following transient expression of a phospho-mimicking EZH2 (T345D) mutant. Input controls (left). mRNA levels of NFKBIZ and its target genes were normalized to RPL37A (right). n = 3 ± SD. (H) Overexpression of IκBζ overrides the inhibitory effects of EPZ6438 (EPZ) on IL-36α–stimulated gene expression in primary keratinocytes. n = 3 ± SD. Significance was calculated using a 1-way ANOVA for multiple groups and a 2-tailed Student’s t test for comparing 2 groups: *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Recombinant human IL-36α (6995-IL; aa 6-158), IL-36γ (6835-IL; aa 18-169), and mouse IL-36α (7059-ML; aa 6-160) were purchased from R&D Systems.

Techniques: Activity Assay, Phospho-proteomics, Western Blot, shRNA, Knockdown, Inhibition, Luciferase, Mutagenesis, Expressing, Gene Expression, Over Expression

Journal: The Journal of Clinical Investigation

Article Title: The CDK4/6-EZH2 pathway is a potential therapeutic target for psoriasis

doi: 10.1172/JCI134217

Figure Lengend Snippet: (A and B) Detection of methylated STAT3 by coimmunoprecipitation. EZH2 and STAT3 (A) or CDK6 and STAT3 (B) were transiently expressed in HEK293T cells. After 1 hour of treatment with (A) abemaciclib (Abe) or (B) EPZ6438 (EPZ), cell lysates were prepared and subjected to immunoprecipitation using a STAT3-specific antibody or control IgG. (C) NFKBIZ promoter-driven luciferase activity in HEK293T cells, transiently expressing CDK6 and EZH2, alone or in combination with WT (wt) STAT3 or methylation-defective STAT3 mutant (K180R). n = 3 ± SD. (D) Analysis of IκBζ and IκBζ target gene expression in STAT3 wt or STAT3 K180R-expressing HaCaT cells. STAT3 wt or STAT3 K180R constructs were transiently expressed in STAT3-KO HaCaT cells, followed by stimulation for 1 hour with IL-36α. n = 3 ± SD. (E) Chromatin immunoprecipitation (ChIP) of STAT3, EZH2, or IgG control in STAT3-KO HaCaT cells reconstituted with either STAT3 wt or STAT3 K180R after 30 minutes of stimulation with IL-36α. Fold enrichment at the NFKBIZ promoter or at the myoglobin genomic region (MB; as negative control) was calculated relative to the IgG control. n = 3 ± SD. (F) ChIP of STAT3, EZH2, CDK4, and CDK6 in IL-36α–stimulated HaCaT cells stimulated for 30 minutes with IL-36α. Shown is the fold enrichment over IgG control. n = 3 ± SD. Significance was calculated using a 1-way ANOVA for multiple groups and a 2-tailed Student’s t test for comparing 2 groups: *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Recombinant human IL-36α (6995-IL; aa 6-158), IL-36γ (6835-IL; aa 18-169), and mouse IL-36α (7059-ML; aa 6-160) were purchased from R&D Systems.

Techniques: Methylation, Immunoprecipitation, Control, Luciferase, Activity Assay, Expressing, Mutagenesis, Targeted Gene Expression, Construct, Chromatin Immunoprecipitation, Negative Control

Journal: The Journal of Clinical Investigation

Article Title: The CDK4/6-EZH2 pathway is a potential therapeutic target for psoriasis

doi: 10.1172/JCI134217

Figure Lengend Snippet: (A) Expression data from skin biopsies of 64 healthy individuals and 58 patients with psoriasis were analyzed from the GEO profile data set GDS4602. Shown are normalized expression values for CCND1, CCND2, and CCND3. EZH2 mRNA (B) and protein levels (C) in human skin samples from healthy individuals and patients with psoriasis; retrieved from the same data set as in A and B. Significance was calculated with a 1-way ANOVA test: *P < 0.05; **P < 0.01; ***P < 0.001. Scale bars: 100 μm. (D) Analysis of Ccnd2, Ccnd3, and Ezh2 mRNA levels in IMQ-treated mice ears at day 6. Values were normalized to Actin. n = 6 per group ± SEM. (E) Analysis of Ccnd2, Ccnd3, and Ezh2 mRNA levels in IL-36α–treated mice ears at day 5. n = 6 per group ± SEM. Significance was calculated using a 2-tailed Student’s t test: *P < 0.05; **P < 0.01; ***P < 0.001. (F) IHC staining of EZH2, cyclin D2, and cyclin D3 in untreated (Ctrl) and IMQ-treated mouse ears at day 6. Scale bars: 40 μm.

Article Snippet: Recombinant human IL-36α (6995-IL; aa 6-158), IL-36γ (6835-IL; aa 18-169), and mouse IL-36α (7059-ML; aa 6-160) were purchased from R&D Systems.

Techniques: Expressing, Immunohistochemistry

Journal: The Journal of Clinical Investigation

Article Title: The CDK4/6-EZH2 pathway is a potential therapeutic target for psoriasis

doi: 10.1172/JCI134217

Figure Lengend Snippet: (A) Ear thickness measurements during topical treatment of mice with IMQ with or without abemaciclib (Abe; 10 μL of a 2% solution) or the EZH2 inhibitor CPI-169 (CPI, 10 μL of a 5% solution). n = 6 mice per group ± SEM. (B) H&E staining of untreated (Ctrl), IMQ-, IMQ and Abe–, or IMQ and CPI–treated ears. Scale bars: 100 μm. (C) Phospho-RB (pRB) and H3K27me3 staining after 6 days of treatment validated effective CDK4/6 and EZH2 inhibition, respectively. Scale bars: 40 μm. (D) Infiltrating immune cells in mouse ears at day 6 of treatment were quantified as follows: Neutrophils: CD45+, CD11b+, Ly6G+; macrophages: CD45+, CD11b+, F4/80+; T cells: CD45+, CD3+, and αβ-TCR+ or γδ-TCR+. n = 3 mice per group ± SEM. (E) Flow cytometry analysis of IMQ-treated or IMQ and CPI-169–treated mouse ears at day 6. (F) Protein levels in untreated (Ctrl) and treated mouse skin tissue at day 6. (G) Ear thickness of IL-36α–treated mice at day 5. Ears of mice were daily treated by intradermal injections with 1 μg IL-36α. Control mice received injections with PBS. Additionally, mice received topical treatment with ethanol as control (Veh), 2% abemaciclib (Abe), or 5% CPI-169 (CPI). n = 6 mice per group ± SEM. (H) H&E staining of PBS- or IL-36α–treated ears at day 5. Scale bars: 100 μm. (I) Immunoblot analysis of IκBζ, EZH2 phosphorylation (pEZH2 T345) and STAT3 activation (pSTAT3 Y705) in treated mouse skin tissue at day 5. pRB and H3K27me3 were analyzed as positive controls for drug action. Significance was calculated using a 1-way ANOVA for multiple groups and a 2-tailed Student’s t test for comparing 2 groups: *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Recombinant human IL-36α (6995-IL; aa 6-158), IL-36γ (6835-IL; aa 18-169), and mouse IL-36α (7059-ML; aa 6-160) were purchased from R&D Systems.

Techniques: Staining, Inhibition, Flow Cytometry, Control, Western Blot, Phospho-proteomics, Activation Assay

Journal: The Journal of Clinical Investigation

Article Title: The CDK4/6-EZH2 pathway is a potential therapeutic target for psoriasis

doi: 10.1172/JCI134217

Figure Lengend Snippet: All analyses were performed with n = 6 mice per group ± SEM. (A) Treatment scheme for the therapy using the IMQ mouse model. To explore whether CDK4/6 and EZH2 inhibitors suppress already-established psoriasis-like skin inflammation, mice were first treated with IMQ, followed by the application of 2% abemaciclib or 5% CPI-169 solution starting at the third IMQ application. (B) Ear thickness measurements during treatment. (C) H&E staining of untreated (Ctrl), IMQ-, IMQ and Abe–, or IMQ and CPI–treated ears. H&E staining shows the prevalence of psoriasis-like symptoms at IMQ day 2 when the inhibitors were applied for the first time. Scale bars: 100 μm. (D) Quantification of infiltrating immune cells in mouse ears at day 6. Immune cell subpopulations were quantified as in Figure 6D. n = 3 mice per group ± SEM. (E) Protein levels in untreated (Ctrl) and IMQ-treated mouse skin tissue in the presence or absence of abemaciclib or CPI-169 at day 6. Mice were treated as in A. FOXM1 and H3K27me3 were analyzed as positive controls for drug action. (F) Treatment scheme in the IL-36–induced psoriasis mouse model. IL-36–mediated psoriasis-like dermatitis was induced by administration of 1 μg IL-36α at every second day. Control mice received PBS. Starting from day 4 of IL-36α injection, ethanol as Vehicle (Veh), 2% abemaciclib, or 5% CPI-169 were daily applied by topical administration. (G) Ear thickness measurements during IL-36α treatment. (H) H&E staining of PBS- or IL-36α–treated ears at day 9. Scale bars: 100 μm. (I) Immunoblot analysis in IL-36α–treated mouse skin tissue at day 9. Significance was calculated using a 1-way ANOVA for multiple groups and a 2-tailed Student’s t test for comparing 2 groups: *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Recombinant human IL-36α (6995-IL; aa 6-158), IL-36γ (6835-IL; aa 18-169), and mouse IL-36α (7059-ML; aa 6-160) were purchased from R&D Systems.

Techniques: Staining, Control, Injection, Western Blot